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Short communication

Detection of Neospora caninum in dog organs using realtime PCR systems

Farida Ghalmi a,c, Bernard China b,d,*, Rachid Kaidi e,Georges Daube b, Bertrand Losson a

aUniversity of Liege, Faculty of veterinary Medicine, Department of Infectious Diseases, Parasitology, Sart Tilman, B43, 4000 Liege, Belgiumb

University of Liege, Faculty of veterinary Medicine, Department of Food sciences, Microbiology, Sart Tilman, B43b, 4000 Liege, Belgiumc National Veterinary School of Algiers, BP 161 Hassen Badi El-Harrach, Algiers, Algeria

dScientific Institute of Public Health, Clinical Biology, rue J. Wytsman 14, 1050 Brussels, Belgium

eUniversity Saad Dahlab of Blida, Veterinary Sciences Department, Blida, Algeria

Received 29 January 2008; received in revised form 3 April 2008; accepted 9 April 2008

Abstract

Neospora caninum is a parasite responsible for paresis in dogs. The dog can harbour enkysted parasites in several organs. The

detection of N. caninum was performed using 3 different real time PCR systems all amplifying the NC5 DNA region. One system

was based on Sybr1green, one on PlexorTM technology and the last on Taqman1 probe. Comparison of the three methods indicated

that the detection limit was 1 equivalent genome on pure DNA but that this detection limit increased in the presence of foreign DNA

using the Sybrgreen and Plexor systems. Therefore, the Taqman system was chosen to detect N. caninum in liver and spleen of

naturally infected dogs. The overall prevalence was 32.2%. Comparison between PCR results and serological results using IFAT

showed that among the 28 PCR positive dogs only 9 were seropositive and that 8 seropositive dogs were PCR negative. Therefore

serology can underestimate the real carriage in dogs. However, PCR methods must be improved in terms of sensitivity and inhibition

problems.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Neospora caninum; Dog; Real time PCR; Organs

1. Introduction

Neospora caninum is a heteroxenous cyst-forming

apicomplexan parasite responsible for diseases in

animals. The major clinical manifestations are hind

limb paralysis in dog and abortion in cattle worldwide

(Dubey et al., 2006). The dog is the definitive host and

the cattle are the intermediate host (Georgieva et al.,

2006). Dogs are infected by ingestion of contaminated

food of bovine origin. In dogs, the parasite can reach

organs such as the brain, spinal cord, retina, muscles,

thymus, heart, liver, kidney, stomach, adrenal gland, and

skin (Peters et al., 2000) where it can form cysts and

persist for a long time leading to chronic disease

(Georgieva et al., 2006). Reactivation of the parasite can

occur when the immune system of the host is depressed

(Georgieva et al., 2006). Serology is the principal

method of N. caninum diagnosis. It includes IFAT

considered as the Gold standard (Dubey and Schares,

2006), ELISA including a lot of variations and

commercially available kits (Dubey and Schares,

2006), DAT (Packham et al., 1998) and Western blot

as a confirmation test. Nevertheless, serology is an

www.elsevier.com/locate/vetpar

Available online at www.sciencedirect.com

Veterinary Parasitology 155 (2008) 161–167

* Corresponding author at: Scientific Institute of Public Health,

Clinical Biology, rue J. Wytsman 14, 1050 Brussels, Belgium.

Tel.: +32 2 642 53 85; fax: +32 2 642 56 45.

E-mail address: [email protected] (B. China).

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2008.04.007

mailto:[email protected]://dx.doi.org/10.1016/j.vetpar.2008.04.007http://dx.doi.org/10.1016/j.vetpar.2008.04.007mailto:[email protected]

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indirect diagnosis test. Moreover, in case of early or

chronic infection with cysts formation, the serology

could be negative even if the parasite is present in the

host. Therefore, direct diagnosis methods can be useful.

These methods include immunohistochemistry, animal

infection or PCR (Dubey and Schares, 2006). PCR has

been demonstrated to be more sensitive to immunohis-tochemistry (Dubey and Schares, 2006). Classical PCR

where the amplification and the detection are separated

was the first technique described in N. caninum

amplification (Payne and Ellis, 1996; Yamage et al.,

1996). Semi-nested or nested PCR was also developed to

increase the sensitivity (Buxtonet al., 1998; Baszler et al.,

1999). The major targets were the locus Nc5, a repetitive

sequence present in the N. caninum genome and the ITS1

locus present in ribosomal DNA operons (Gondim et al.,

2004). Real time PCR systems using a specific Taqman

probe (Collantes-Fernandez et al., 2002) or hybridisationprobes (Müller et al., 2002) were also described. In this

study, the Nc5 sequence was selected since the ITS1 is

potentially more variable (Gondim et al., 2004). A

sensitive detection method of N. caninum in dog organs

by real time PCR using either specific Taqman1 probe,

PlexorTM or Sybr1green systems was described.

2. Materials and methods

2.1. Reference material

N. caninum strain NC-1 (Dubey et al., 1988) was used

to produce reference DNA. Briefly, NC-1 tachyzoites

(ATCC50843; Dubey et al., 1988) were used to infect

vero cells (ATCC CRL-1587). The growing tachyzoites

led to the vero cells lyses and the tachyzoites were

withdrawn. The total content of the culture flask was

centrifuged (1500 g for 20 min) and the pellet were

resuspended in phosphate buffered saline (PBS, pH 7.4)

and flushed through a 25 gauge needle in order to lyse to

vero cells. The tachyzoites were harvested by centrifuga-

tion (1500 g for 20 min). The pellets were resuspended

in PBS (pH 7.4) and the number of tachyzoites in thesuspension was determined using a haemocytometer.

2.2. Indirect fluorescence antibody test

The IFAT was performed as previously described

(Dubey et al., 1988). Briefly, 104 tachyzoites per well

were used to coat a 10-wells tefloned slide. The slides

were washed and 20 ml of diluted sera (1/50–1/800)

were added to the wells for 25 min at 37 8C. After

washing, a FITC-conjugated anti-dog IgG (Sigma–

Aldrich, St Louis, USA) was added and incubated as

above. After washing, the slides were mounted and

positive tachyzoites were looked for under epifluores-

cence microscope (400).

2.3. Organs

From November 2004 to June 2005, 87 dogs from adog pound in Algiers were euthanised by pentobarbital

overdose and the liver, the spleen and the mesenteric

lymph nodes were removed by surgery. The samples

were conserved transported to the lab on ice in a cooling

bag. In the lab, the organs were stored at 20 8C till

DNA extraction. Moreover, none of the studied dogs

showed neosporosis symptoms.

2.4. DNA extraction

The DNAwas extracted from 50 mg of each organ orfrom NC-1 tachyzoitzes (4 106 ml1) using a phenol

chloroform based method (Sambrook et al., 1989). The

organs were incubated in a lysis buffer (Tris 10 mM,

EDTA 100 mM, pH 8, SDS 0.5%) containing proteinase

K (0.1 mg ml1) at 56 8C for 18 h. The lysate was

extracted twice with a phenol/chloroform/isoamylic

alcohol (25/24/1, v/v/v) solution. The DNA was

precipitated with ethanol in the presence of 3 M sodium

acetate. After centrifugation (13,000 g for 30 min),

the pellets were washed with 70% ethanol. The final

DNA pellets were resuspended in 200 ml Tris EDTAbuffer. The DNA concentration was spectrophotome-

trically estimated (Nanodrop 100, Isogen, The Nether-

lands). Finally the DNA was stored at 20 8C until use.

For NC-1 DNA extraction, the DNA was extracted using

the same protocol as above on 1 ml of pelleted

tachyzoites but the lysis time was reduced to 3 h.

2.5. PCR systems

The primers were selected on base of the NC5

sequence (Genbank accession number X84238). For the

different real time PCR systems, the amplificationswere performed on ABI7000 (Applied Biosystems,

Foster City, CA, USA). The PCR efficiency has been

calculated by using the slope of the standard curve

E = (1 101/slope) 100.

2.6. Sybrgreen system

For the Sybrgreen real time system the primers were

modified from NP7-NP4 system (Baszler et al., 1999)

using the Oligo6 software (Medprobe, Oslo, Norway).

Upper primer: 561U20: GGGAGTTGGTAGCGGT-

F. Ghalmi et al. / Veterinary Parasitology 155 (2008) 161–167 162

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GAGA and lower primer: 806L20: GCCTCCCAATGC-

GAACGAAA. The optimal annealing temperature was

59.9 8C, the expected amplicon Tm was 89.7 8C and the

amplicon size was 265 bp as calculated by Oligo6

software (Medprobe, Oslo, Norway). The PCR mix was

12.5 ml of Itaq Sybrgreen mix 2 (BioRad, Nazareth,

Belgium), 0.25 ml of each primer (50 mM), 5 ml of targetDNA and PCR grade water to 25 ml. The cycles were:1

(50 8C for 2 min), 1 (95 8C for 3 min.), 50 (95 8C for

15 s, 60 8C for 1 min). The amplification reaction was

followed by a melting curve (from 60 to 95 8C).

2.7. Plexor system

For Plexor system the primers were selected using the

Plexor primer design software (www.promega.com/

PlexorTM resources/ ). The primers were purchased

from Eurogentec (Seraing, Belgium). NC5PLEX: 6-FAM-ACAGAACACTGAACTCTGGATAAGTATCA

and NC5FRIEND: GGATACGTGGTTTGTGGTTAGT-

CATTC. The Tm and the size of the amplicon were

83.2 8C and 101 bp, respectively, as calculated by the

Oligo6 software. The primers were resuspended together

in MOPS EDTA buffer at a concentration of 5 mM using

buffer purchased from the Plexor qPCR system

(Promega, Madison, WI, USA). The primer stock

solution was stored in dark. The reaction mix was

12.5 mM Plexor master mix 2, 1 ml of primers solution

(5 mM) and 6.5 ml of nuclease free water. Theamplification was performed from 5 ml target DNA.

The thermal cycling program was 1 (95 8C for 2 min)

and50 (95 8C for 5 s, 60 8C for 35 s). The amplification

reaction wasfollowedby a dissociation protocol.The raw

data of amplification (D Rn) and dissociation were

exported to the Plexor analysis software (available for

download at: www.promega.com/PlexorTM resources/ ).

2.8. Taqman system

The primers and probes were selected using the

Primer Express software (Applied Biosystems, FosterCity, CA, USA). NC5-550: GGGTGAACCGAGG-

GAGTTG, NC5-596: ACGTGAGGAATGACTAAC-

CACAA, NC5 probe: FAM-AGCGGTGAGAGGT-

GGGATACGTGG. The Primers stock solution was

50 mM and the probe stock solution was 20 mM. The

real time PCR mix was: 12.5 ml of qPCR master mix 2

(Eurogentec, Seraing, Belgium), 0.25ml of each primer,

0.25 ml of NC5 probe, 5 ml of DNA, and 6.75 ml of

nuclease free water. The amplification thermocycling

was: 1 (50 8C for 2 min), 1 (95 8C for 10 min), and

50 (95 8C for 15 s, 58 8C for 1 min).

Moreover an internal PCR control was constructed

using the hsp60 gene of Bifidobacterium thermophilum

since a probe was already available in the lab. The gene

was amplified using hybrid primers: Cineoup: GGGT-

GAACCGAGGGAGTTG-TGTGGAGACCAAAGGA-

CCAGAT and Cineodwn: ACGTGAGGAATGACT-

AACCACAACATCCTGGCCGACCTTGT. The DNAof B. thermophilum (ATCC25866) was amplified by

classical PCR using the following mix: 2 ml of target

DNA, 0.2 ml of fideliTaq (usb, Cleaveland, OH, USA),

2 ml of 10 buffer, 0.2 ml of each primers (40 mM),

dNTPs 2.5 mM 1.6 ml; 13.8 ml nuclease free water. The

amplification thermocycling was performed in a mas-

tercycler gradient (Eppenddorf, Hamburg, Germany) and

consisted of 1 (94 8C for 5 min), 40 (94 8C for 30 s,

58 8C for 30 s, 72 8C for 60 s), 1 (72 8C for 5 min) and

keep at 4 8C. A tenth of the amplification product was

reamplified in the same condition using the primers NC5-550andNC5-596. The obtained 142 bp PCR product was

purified (Qiaquick PCR purification kit, Qiagen, Venlo,

The Netherlands) and decimally diluted. The lowest

amplifiable IPC concentration was determined by real

time PCR using NC5-550 and NC5-596 as primers and

CIprobe: NED-ACGATTTCCGCTGCT-MGB as probe.

The mix was 12.5 ml of qPCR master mix 2

(Eurogentec, Seraing, Belgium), 0.25 ml of each primer,

0.25 ml of CI probe, 5 ml of DNA, 6.75 ml of nuclease-

free water. The thermocycling was performed as above.

The selected IPC dilution was 10

18

with an average Ctvalue of 42.44. For N. caninum from dog organs the PCR

mix was: 12.5 ml of qPCR master mix 2 (Eurogentec,

Seraing, Belgium), 0.25 ml of each primer, 0.25 ml each

probe, 2 ml ofIPC, and 6.5 ml of nuclease free water. The

DNA amplification was performed on 5 ml DNA.

2.9. DNA sequencing

The PCR product was purified (Qiaquick purification

kit, Qiagen, Venlo, The Netherlands) and sequenced

(DYEnamic ET Dye Terminator Cycle Sequencing Kit,

Amersham Biosciences, Diegem, Belgium) using thePCR primers. The sequencing products were analysed

by capillary electrophoresis (MegaBACE 500, Amer-

sham Biosciences, Diegem, Belgium).

3. Results and discussion

3.1. PCR systems comparison

Beside the Sybrgreen (Schneeberger et al., 1995) and

Taqman (Holland et al., 1991) systems a new

technology, the Plexor system, has been tested (Jonhson


http://www.promega.com/PlexorTM%20resources/http://www.promega.com/PlexorTM%20resources/http://www.promega.com/PlexorTM%20resources/http://www.promega.com/PlexorTM%20resources/http://www.promega.com/PlexorTM%20resources/http://www.promega.com/PlexorTM%20resources/

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et al., 2004; Sismour et al., 2004). This technology use a

primer labelled with a fluorescent reporter and a modified

base (iso-dC) at the 50 end. As amplification proceeds,

fluorescence is reduced by site-specific incorporation of

iso-dG tagged with the fluorescent quencher dabcyl

opposite the iso-dC base in the modified primer. The

close proximity of the dabcyl to the fluorescent reporterresults in a reduction of the fluorescent signal. Therefore

in the Plexor approach, contrary to the two others

technologies, the fluorescence decreases during the

amplification. As in Sybrgreen technology, a dissociation

curve was carried out after the amplification in order to

check the amplification specificity by the amplicon

fusion temperature determination. These three technol-

ogies were used to detect N. caninum DNA. First of all,

the different PCR systems have been tested on pure

tachyzoites DNA. The NC-1 DNA was extracted from

4 106 tachyzoites and resuspended in 200 ml t o a

concentration of 2 104 equivalent genome per ml. The

PCR were performed on 5 ml of serial dilution of this

suspension. The amplification reactions contained 105,104, 103, 102,101 and 100 equivalent genomes, respec-

tively (Fig. 1). All the systems were able to detect one

equivalent genome. For Plexor and Sybrgreen systems,

the specificity of the amplification has been checked

using a dissociationcurve (Fig.1). Since theobserved Tm

was slightly lower than the expected Tm, the specificity

of the amplification was positively confirmed by


Fig. 1. PCR systems on pure N. caninum DNA. PCR have been performed from 107 (7), 106 (6), 105 (5), 104 (4), 103 (3), 102 (2), 101 (1), 100 (0)

equivalent genomes of NC-1 N. caninum DNA. (A) Dissociation curve (dF /dT ) in function of the temperature (8C) for sybrgreen amplifcation. The

melting temperature of the amplicons was 86.1 8C. Standard curve for Sybrgreen (B), Plexor (D) and Taqman (F) amplification, respectively. Ct

values in function of the logarithm of DNA copies number (log N ). The curve equation is indicated with the determination coefficient ( R2) and the

PCR efficiency (E). (C) Dissociation curve (dF /dT ) in function of the temperature (8C) for Plexor amplification. The melting temperature of the

amplicons was 79.9 8C). (E) Taqman amplification curves. The fluorescence in function of the cycle number. The horizontal line corresponds to the

fluorescence threshold.

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amplicon sequencing (data not shown). Nevertheless, the

Taqman system was the only system were the PCR

efficiency could be truly calculated on the all DNA

dilutions as for the two other systems, the measured

fluorescence was due to the specific amplification and

primers dimers (Fig. 1). Therefore the PCR efficiency

was only calculated on the 5 first dilutions for Plexor andSybrgreen system. The calculated PCR efficiency was

100% for Taqman system and 97% for Sybrgreen and

Plexor systems (Fig. 1). Since the aim was to amplify N.

caninum DNA fromdog tissues, the following step was to

estimate the detection limit when N. caninum DNA was

mixed with organ DNA. Therefore, N. caninum DNAwas

mixed with chicken liver DNA (100 mg ml1) in order to

obtain 0, 1, 10, 1000, 10,000, and 100,000 equivalent

genome in 500 ng of liver DNA. The different real time

PCR was performed (Fig. 2). Only the Taqman PCR

systemwas able to detect one equivalent genome but with

a Ct value of 45.53 (0.4). In Plexor and Sybrgreen

systems, the detection limit was 10 equivalents genome

per PCR reaction. It is most probably related to the higher

specificity of the Taqman amplification due to the

presence of the specific probe allowing to fish the rightamplicon even among non-specific amplicons. The two

other techniques are more affected by non-specific

amplification especially when the amount of target DNA

was low. In these conditions, the peak corresponding to

the specific amplicon was difficult to identify. More fine

tuning is probably required to increase the specificity of

the amplification. Therefore, the Taqman approach was

selected for the detection of N. caninum in organs of

naturally infected dogs.


Fig. 2. PCR systems on N. caninum DNA mixed with foreign DNA. PCR have been performedfrom 107 (7), 106 (6), 105 (5), 104 (4), 103 (3), 102 (2),

101 (1), 100 (0) equivalent genomes of NC-1 N. caninum DNA. (A) Dissociation curve (dF /dT ) in function of the temperature (8C) for sybrgreen

amplification. The melting temperature of the amplicons was 86.2 8C. Primer dimers were predominant for 100 equivalent genome (0). Standard

curve for Sybrgreen (B), Plexor (D) and Taqman (F) amplification, respectively. Ct values in function of the logarithm of DNA copies number

(log N ). The curve equation is indicated with the determination coefficient ( R2) and the PCR efficiency (E). (C) Dissociation curve (dF /dT ) in

function of the temperature (8C) for Plexor amplification. The melting temperature of the amplicons was 80.0 8C. Primers dimers were present from

102 to 100 equivalent genomes and the primers dimers curve was predominant for 100 equivalent genome amplification. (E) Taqman amplification

curves. The fluorescence in function of the cycle number. The horizontal line corresponds to the fluorescence threshold.

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Since DNA isolated from organ can contain PCR

inhibitors, the construction of an internal PCR control

(IPC) was performed. The addition of a competitor

molecule in the PCR mix could lower the detection limit

of the system. In order to check this, the previous

experiments were reproduced including the IPC. The

detection limit was not modified but the measured Ctvalues were significantly increased (46.29 0.3;

p < 0.01).

3.2. Dog organs

A prevalence study was performed on 87 dogs of the

central pound of Algiers. The liver and the spleen was

analysed on each animal using the Taqman real time

PCR system. Out of the 87 dogs, 59 (68%) were

negative and 28 (32%) were positive in at least one

organ. Among the 59 positive, 14 (50%) were positivein both organs, 11 (12.4%) were positive only for the

spleen sample and 3 (3.4%) were positive for the liver

sample. It is probably due to the sampling since the

DNA extraction was only performed on 50 mg of organ.

Moreover, liver is a bigger organ than spleen therefore

the sample represented a larger proportion of the whole

spleen than of the whole liver. It is maybe possible to

increase the sensitivity of the method by increasing the

size of the sample and by decreasing the elution volume

of the DNA. Nevertheless, the PCR can only be

performed on a small volume (here 5 ml) that is also alimitation in the sensitivity of the method. But, some

dogs (14/87, 16%) were positive for both organs

indicating a big colonisation of the organs by N.

caninum. Another point that can decrease the efficiency

of the PCR was the presence of PCR inhibitors. Out of

87 tested samples, 10 showed inhibition. The inhibition

was solved by DNA dilution, but this lead to a decrease

of the sensitivity. After the dilution, 2 samples became

positive and 8 became negative. The DNA extraction

method could be improved to eliminate the inhibitors.

Finally, the two selected organs were liver and spleen

even thought the major site of N. caninum is the brain(Barber and Trees, 1996). Therefore, it is probable that

this study underestimated the prevalence of N. caninum

due to the organ choice.

3.3. Quantification

Since real time PCR is a quantitative method the

number of parasite per gram of organ can be

estimated. Using NC1 DNA dilution, a standard

curve was constructed and a quantification of the

number of parasite present in the organ was

attempted. The Ct value from organs ranged from

34.70 to 43.40 corresponding a range of 1 to 102.6

genome equivalents by PCR reaction or from 800 to

4 105 parasites per gram of organ. The results

indicated that the level of contamination varied

widely between dogs. But it was just an estimation

based on the principle that the parasites wererandomly distributed in the organ.

3.4. PCR versus IFAT

A blood sample was taken before euthanasia of the

dogs and the serum was isolated. Specific N. caninum

antibodies were detected using IFAT. Out of 87 dogs, 28

(32.2%) were positive. The real time PCR and the IFAT

results were compared. The relative accuracy was 69%

(60/87). When the IFAT was considered as the reference

method, the relative sensitivity was 53% (9/17) and therelative specificity was 73% (51/70). The kappa value

was 0.21 considered as bad agreement (Winepiscope

2.0; http://infecepi.unizar.es/ratio/soft_sp.htm). Dogs

positive for PCR and negative for IFAT were probably

due to the fact that in chronic infection the parasite

forms cysts in the organs that stimulate slightly the

immune system. More surprisingly, 8 dogs were

positive by IFAT and negative by PCR indicating that

PCR was less sensitive than IFAT. Among these dogs

were 4 dogs showed a PCR inhibition. These four dogs

showed an IFAT titre of 1/800. The 4 other dogs wereonly slightly positive by IFAT with a titre of 1/200. It

indicated either than the PCR sensitivity was too low

mostly when dilution was performed due to the

inhibition or that the dog was seropositive but that

the parasite was not present in tested organs. Never-

theless, the major advantage of PCR on serology is to

directly detect the presence of the parasite. The major

disadvantage is the incapacity to perform the test on

living dogs. The positive PCR results obtained on cattle

blood opened an interesting way for PCR testing on

living animals (Okeoma et al., 2004). Even if the

sensitivity of the method must be increased, real timePCR offers an interesting alternative to the histological

methods for the detection of N. caninum in dogs’

organs.

Acknowledgements

FG thanks the Algerian Ministry of Higher Teaching

to contribute to her PhD formation. FG was financially

supported by the Belgian Technical Cooperation fro this

work. The authors thank Dr E. Kissling for her critical

reading of the manuscript.


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